Insulin treatment of fat and muscle cells causes a rapid increase in the rate of glucose transport into these cells. This effect is a major contributor to the lowering of the blood glucose level by insulin. The basis of the effect is the movement to and fusion of specialized intracellular vesicles containing the glucose transporter GLUT4 with the plasma membrane. This process is known as GLUT4 translocation. Our overall goal is to elucidate at the molecular level how GLUT4 translocation occurs. The main focus in the coming grant period is to describe in detail how signaling from the insulin receptor causes GLUT4 translocation in cultured adipocytes. Insulin activates the serine protein kinase Akt, and considerable evidence indicates that Akt activation initiates GLUT4 translocation. In the previous grant period we and others obtained evidence indicating that Akt activation elevates the GTP form of the small G protein Rab10 and that Rab10 in its GTP form is required for GLUT4 translocation. In addition, there is evidence indicating that besides the effect on Rab10, there is another signaling input to GLUT4 translocation downstream of the protein kinase Akt. This input may stimulate the rate at which GLUT4 vesicles tethered to the plasma membrane fuse with it. We have two specific aims that build upon these results. The first aim is to find and characterize the effector proteins for Rab10-GTP participating in GLUT4 translocation. We have identified two likely effector proteins, each of which binds directly to Rab10-GTP. One is Sec15B, a component of the exocyst, which is a complex that tethers vesicles to membranes prior to fusion. The other is the EH domain binding protein 1, a protein previously established to be required for GLUT4 translocation. The second aim is to characterize a group of six proteins that are candidates to be the key component(s) of the second signaling input to GLUT4 translocation. We recently discovered these proteins through a comprehensive mass spectral analysis of the phosphorylation sites on the proteins in the plasma membranes of unstimulated and insulintreated adipocytes. Each candidate protein undergoes insulin-stimulated phosphorylation and has properties expected for a protein that may regulate the fusion of GLUT4 vesicles with the plasma membrane. We will determine whether phosphorylation of one or more of these proteins is required for GLUT4 translocation, and if so, how it is. This research is directly relevant to diabetes. Detailed knowledge of GLUT4 translocation may lead to better therapeutic regimes or agents for both type 1 and type 2 diabetes.